Human immunodeficiency virus protease (HIV PR) is an important target for the inhibition of viral replication. Though many potent in vitro inhibitors have been developed, most of them are either inactive or toxic in vivo, or mutant forms of the virus emerge which are resistant. (See J. H. Condra, et al., Nature (1995): vol. 374, 569-571; M. Markowitz, et al., J. Virol. (1995): vol. 69, 701; D. J. Kempf, et al., Proc. Natl. Acad. Sci. USA (1995): vol. 92, 2484-2488; A. K. Ghosh, et al., J. Med. Chem. (1994): vol. 37, 2506-2508; P. Y. Lam, et al., Science (1994) vol. 263, 380-384; N. A. Roberts, et al., Science (1990): vol. 248, 358; and E. E. Kim, et al., J. Am. Chem. Soc. (1995): vol. 117, 1181-1182.)
The lack of animal systems to test the efficacy of the inhibitors further slows down the drug development process. Recently a similar protease has been identified in the life cycle of feline immunodeficiency virus (FIV). (R. L. Talbott, et al., Proc. Natl. Acad. Sci. USA (1989): vol. 86, 5743-5747; and N. C. Pedersen, et al. Science (1987): vol. 235, 790-793.) FIV is a virus which leads to clinical symptoms comparable to those observed in human acquired immune deficiency syndrome (AIDS). Studies have shown that up to 14% of the cats surveyed in the USA and Canada are infected with FIV. (J. K., Yamamoto, et al., JAVMA (1989): vol. 194, 213-220.) In Japan, the figure is 28.9%. (T. Ishida, et al., JAVMA (1989): vol. 194, 221-225.
In drug resistant mutants of HIV, there are at least six cases where HIV PR residues mutate to the structurally aligned residue found in FIV PR. The amino acid changes in HIV PR are V32I (137-FIV), L90M (M107-FIV), NB8D (D105-FIV), 150V (V59′-FIV), K20I (I25-FIV), and Q29K (K109-FIV). For a list of resistant HIV PR mutants is disclosed by J. W. Mellors, et al. (International Antiviral News (1995): vol. 3, 8-13.) The structure alignment was derived from the X-ray crystal structure of FIV PR solved by A. Wlodawer (reference 36). Superimposition of the two proteases based on their X-ray structures indicates similarities between the two proteases and the drug resistant HIV proteases.
HIV PR is a 99 amino acid aspartyl protease which functions as a homodimer. (M. A. Navia, et al., Nature (1989): vol. 337, 615-620; and D. D. Loeb, Virol. (1989): vol. 63, 111-121.) FIV PR is also a homodimeric aspartyl protease which consists of 116 amino acid residues. (J. H. Elder, Infectious Agentsand Disease (1994): vol. 2, 361-374.) Both HIV and FIV proteases are responsible for the processing of viral gag and gag-pol polyproteins into structural proteins and enzymes essential for the proper assembly and maturation of full infectious virions. (S. K. Thompson, Bioorg. Med. Chem. Lett. (1994): vol. 4, 2441-2446.) In particular HIV and FIV proteases show high specificity for the selective cleavage of the Tyrosine/Phenylalanine-Proline amide bonds in the Matrix-Capsid domain of the gag-pol polyproteins, a specificity not exhibited by mammalian cellular proteases which are not known to efficiently hydrolyze peptide bonds involving the proline nitrogen. (C. Debouck, Aids Research and Human Retroviruses (1992): vol. 8, 153-164; and J. H. Elder, J. Virol. (1993): vol. 67, 1869-1876.) It is this specificity that makes HIV PR an attractive target for inhibition. A comparison of the amino acid sequence about the matrix capsid cleavage site (Tyrosine˜Proline bond) in both HIV and FIV is provided below. As can be seen the residues about the cleavage site are the same at four positions, P3, P1, P1, and P2. It is disclosed herein that, due to these similarities, certain HIV PR inhibitors also inhibit FIV PR.
P4P3P2P1P1′P2′P3′P4′HIVPRSerGlnAsnTyr˜ProIleValGlnFIVPRProGlnAlaTyr˜ProIleGlnThr
Activated ketones, in general, have been shown to inhibit most kinds of proteases (Barrett et. al., Proteinase Inhibitors; Research monographs in cell and tissue physiology; Dingle, J. T., Gordon, J. L., General Eds.; Elsevier Science Publishers; Amsterdam, 1986). In particular, a recent study shows the design of three different classes of activated ketones which inhibit the aspartyl protease, renin. These potent analogs display IC50 values from 4000 nM to 4.1 nM and include 1,1,1-trifluoromethyl ketones, α-keto esters, and α-diketones as the activated ketone functionalities (Patel et. al. J. Med Chem. 1993, 36, 2431).
The α-keto-amide core structure is isosterically analogous to the activated ketones but is more potent than the reported hydroxyethylamine or phosphinic acid HIV protease inhibitors that are mechanism-based isosteric core structures.
The α-keto-amide core structure has been used in inhibitors of various enzymes which include serine and cysteine proteases, hydrolases and aminopeptidases. As an example, a series of dipeptidyl and tripeptidyl α-keto amides have been synthesized and evaluated as potent inhibitors for cysteine proteases which include enzymes calpain I, calpain II, cathepsin B, and papain (Li et. al. J. Med. Chem. 1993, 36, 3472). Another study has identified α-keto amide analogs as inhibitors of an epoxide hydrolase (Wong et. al. J. Med. Chem. 1993, 36, 211). Additionally, the inhibition of arginyl aminopeptidase (Ki=1.5 uM), cytosol aminopeptidase (K1=1.0 μM) and microsomal aminopeptidase (K1=2.5 μM) has been observed from α-keto amide analogs which can be derived from 3-amino-2-oxo-4-phenylbutanoic acid amides. (Rich et. al. J. Med Chem. 1992, 35, 451).
The activity of dipeptide isosteres is often enhanced by addition of amino acid residues to either the N and C-terminus of the isostere to improve binding in the active site. The prior art provides examples of such inhibitors which include renin, aspartyl proteases and procine pepsin inhibitors (Rich et. al. J. Med Chem. 1992, 35, 451). The resulting inhibitors generally exhibit high binding affinity to HIV protease. They display, however, instability and/or poor oral bioavailability.
The related art has provided examples of α-keto amide inhibitors of aminopeptidases which contain an unsubstituted proline moiety in the molecule. In particular, Gordon and co-workers have described α-keto amide inhibitors with an unsubstituted proline ring for the themetalloprotease angiotensin converting enzyme (ACE) (Gordon et. al. Biochem. Biophys. Res. Commun. 1984, 124, 141). Additionally, Arai et. al. (Chem. Pharm. Bull. 41, 9, 1583) have reported potent inhibitory activity by a prolyl endopeptidase (PEP) inhibitor (N-[N-(4-phenylbutanoyl)-L-prolyl]pyrrolidine).
What is needed are combinatorial libraries of HIV and FIV protease inhibitors and simple synthetic methods for making same.
What is needed is a class of HIV and FIV protease inhibitor having enhanced possibilities of variability at the P1, and P1′ positions for improving the binding between the enzyme and its inhibitor.
What is needed are methods for screening combinatorial libraries of HIV and FIV protease inhibitors for identifying candidates having both clinically useful inhibitory activity and a potential resistivity to a loss of inhibitory activity due to development of resistant strains of HIV.
What is needed are new HIV and FIV protease inhibitors having clinically useful inhibitory activity and a resistivity to a loss of inhibitory activity due to development of resistant strains of HIV.